Purpose:
Corneal sensory nerves contribute trophic factors to maintain the integrity and function of the cornea and particularly of the epithelium. Little is known whether corneal epithelium is mutually important for the maintenance of corneal nerve function. Herein, we test the possibility that impaired stratified corneal epithelium due to ablation of Shp2 may cause failure of corneal innervation during development and nerve degeneration in adult.

Methods:
Krt14-rtTA/tet-O-Cre/Shp2f/f/Thy-1-YFP quadruple transgenic mice administrated without (referring to Shp2 wild-type) or with (referring to Shp2 null) doxycycline (Dox) from embryonic day 7 (E7) to E12.5 and E15.5, from postnatal day 1 (P1) to P21, and from P21 to P33, respectively. Mouse excised corneas were subjected to whole-mount histology examination under confocal microscopy. Thy-1-YFP was served as a fluorescent marker for comparing the pattern of peripheral corneal innervation with or without Shp2 expression in the corneal epithelium. Transmission electron microscopy (TEM) was also used to examine detail morphology of the corneal nerves.

Results:
YFP signal was first detected from four quadrants of the eye at E12.5 and they covered the entire corneal surface at E16.5 of the control mice, whereas Shp2 conditional knockout in K14+ cells rendered the failure of corneal innervation during embryonic development. Interestingly, Shp2 ablation from fully stratified corneal epithelium at P21 resulted in the degeneration of corneal nerve bundle at P33. TEM examination revealed that individual nerve fibers and nerve bundles wrapped around the basal epithelial cells and the wing cells and the large single fibers contain many mitochondria, clusters of glycogen particles, and vesicles of different types and sizes of the wild-type cornea. In contrast, however, little of such was found in the Shp2 null cornea.

Conclusions:
These data suggest that formation of stratified corneal epithelium is indispensable for corneal innervation during development and maintenance of nerve integrity in adults, and Shp2-mediated receptor tyrosine kinase signaling may play a pivotal role in this corneal epithelium-nerve interaction which is paramount for the corneal morphogenesis and homeostasis.